Arsenic is classified as a carcinogen by the U.S. Environmental Protection Agency. Arsenicals and antimonials are still the first-line drugs for the treatment of sleeping sickness and leishmaniasis, and resistance to the pentavalent antimonial drug Pentostam is becoming a major clinical problem. More recently arsenite (Trisenox) has been approved for use as a chemotherapeutic agent for the treatment of acute promyelocytic leukemia. The goal of this research is the elucidation of the molecular mechanisms in members of two families of arsenate reductases. The arsenate (As(V)) reductase found in many bacteria is typified by the ArsC enzyme encoded by the (ars) operon found on the clinically-isolated E. coli resistance plasmid R773. Eukaryotes have pathways for arsenic detoxification that arose through convergent evolution. The ACR2 gene of Saccharomyces cerevisiae encodes an arsenate reductase required for arsenate resistance in yeast but is not an ArsC homologue. Specific aims include: 1) Structure function relationships in the arsenate reductases: Residues in the E. coli ArsC and S. cerevisiae Acr2p involved in function will be identified such as anion binding sites and residues involved in catalysis. 2) Interaction of ArsC with glutaredoxin. Glutaredoxin 2 (Grx2) is the most efficient hydrogen donor for ArsC. The contact points between Grx2 and ArsC will be determined by a combination of biochemical and genetic approaches including crystallography, chemical crosslinking, surface plasma resonance and NMR. Similar methods will be applied to the interaction of Acr2p and glutaredoxin. 3) The recently identified Leishmania major LmAcr2p will be characterized. The electron donor(s) for LmAcr2p homologue will be determined. Its role in resistance to the Sb(V)-containing drug Pentostam will be examined. The ability of this and other arsenate reductases to reduce Sb(V) will be determined. 4) The structure of eukaryotic arsenate reductases will be determined by x-ray crystallography. 5) Identification of other eukaryotic arsenate reductases from arsenic extremeophiles: Plants and fungi have developed arsenic detoxification systems that allow them to survive in extreme environments. We propose to study the mechanisms of arsenate resistance in two such organisms. One is an arsenate hyper-resistant Aspergillus from the Rio Tinto in Spain, where the effluent from ancient Roman mine still pollutes the river. This fungus can grow in 0.2 M arsenate and has increased rates of arsenate reduction. A second organism is an arsenate hyper-accumulating hyper-resistant fern, Pteris vittata, isolated from the grounds of a Florida lumber company that treats wood with the preservative chromated copper arsenate (CCA). This plant grows in high concentrations of arsenate and reduces it to arsenite, which is accumulated in vacuoles. [unreadable] [unreadable]